This invention pertains to antibacterial compositions and a method of inhibiting bacterial β-lactamases. The prevention and/or treatment of infections in a subject may be accomplished by administering a β-lactamases inhibitor composition that consists of double stranded deoxyribose nucleic acid (DNA) fragments. More specifically, a method of inhibiting a β-lactamases in bacterium includes: contacting the bacterium with an isolated double stranded DNA. In a preferred embodiment, the double stranded DNA is at least 90% identical to SEQ ID No.: 4.
Selecting a site of action is one of the most important decisions made when developing antibacterial compounds. β-lactam antibiotics targets cell wall development, which is one of the most accessible processes of the cell. In 1929, Sir Alexander Fleming indirectly came across these compounds that were produced as a defense mechanism by the fungus Penicilium notatum. Since that time β-lactam antibiotics have become some of the most prescribed chemotherapeutic compounds (Maugh, 1981).
β-Lactam antibiotics include penicillins, cephalosporins, monobactams and carbapenems. These compounds are analogs of peptidoglycans, which are essential to the production of the cell wall. The DD-peptidases (D-alanyl-D-alanine carboxypeptidases/transpeptidases) are the target enzymes of the β-lactam antibiotics. These enzymes catalyze the cross-linkage of peptidoglycans during bacterial cell wall biosynthesis. β-lactam antibiotics form a stable covalent acylenzyme complex that has a much longer half-life than that which is formed with the peptidoglycans. This disrupts the synthesis of the cell wall, which eventually leads to cell death (Kelly et al., 1988; Ghuysen, J. M., 1988). Inhibition of cell wall synthesis proves to be a very effective method in disrupting bacterial cell growth. The lack of a cell wall in mammalian cells is paramount because even at high concentrations of the β-lactam antibiotics, mammalian cells are not affected (Maugh, 1981).
The general structures of penicillin and cephalosporins are shown in FIG. 1. When compared to the structure of peptidoglycans, the ring structure of the β-lactam places strain on the adjacent atoms to have a similar configuration.
β-Lactamases (β-lactamhydrolyases, EC 3.5.2.6) are enzymes that very efficiently catalyze the hydrolysis of the β-lactam ring antibiotics, causing them to lose their bactericidal activity (Fisher et al, 1981, Maugh, 1981) (FIG. 2).
Bacteria that obtain a gene for producing β-lactamases become resistant to β-lactam antibiotics. Many different bacteria have one of these genes including Bacillus cereus, Bacillus anthracis, Bacillus fragillis, Escherichia coli, Bacteriodes, Staphlococcus epidermidis, Streptococcus, Psuedomonas aerugenosa, Providencia, Haemophilus, Xanthomonas maltophilia, Acinetobactor, Citrobactor, Enterobactor, and Branhamella (Danziger and Pendland, 1995). β-Lactamases can be transferred between bacteria when the gene is present on a plasmid. These enzymes are classified based on their primary structure and catalytic properties. Currently there is a four-class system for β-lactamases consisting of A, B, C, and D (Ambler, 1980; Ambler et al., 1991; Joris et al., 1991; Frere, 1995). Those enzymes belonging to classes A, B, and D are all serine-active-site enzymes that resemble serine proteases. During the reaction an acyl-enzyme intermediate is formed with the active site serine leading to the hydrolysis of the β-lactam antibiotics (Rahil and Pratt, 1991). Class B β-lactamases are distinct from the other three classes of β-lactamases. They are referred to as metallo-β-lactamases due to presence of at least one divalent metal ion in the active site for enzymatic activity (Ambler, 1980; Abraham and Waley, 1979). One or two zinc ions are present in all of the native metallo-β-lactamases isolated (Carfi et al, 1995; Concha et al., 1996).
An increasing number of bacteria are obtaining the gene structure necessary to produce β-lactamase, which makes them resistant to β-lactam antibiotics. Understanding the mechanism of hydrolysis of the β-lactam antibiotics is necessary for the production of new antibiotics and inhibitors (Abraham and Waley, 1979; Brenner and Knowles, 1984). For many years chemical alterations to existing β-lactam antibiotics have been used to stay ahead of drug resistance. In fact, cephalosporins have gone through four generations of this process (Maugh, 1981; Pitout et al., 1997). It has become increasingly common for a combination of inhibitor and antibiotic to be used to combat bacteria that are resistant. However, there are a limited number of alterations that can be made to the molecule and still maintain its bactericidal characteristics. The discovery of new classes of antibiotics and β-lactamase inhibitors is a costly and time-consuming process. For these reasons it is important to develop new β-lactamase inhibitors.
Combinatorial chemistry has become a commonly used technique for developing large numbers of possible ligands to target molecules. One technique involving the use of large pools of oligonucleotides for screening of functionality, which was independently developed in the labs of G. F. Joyce (La Jolla) (1989), J. W. Szostak (Boston) (Ellington, A. D. and Szostak, J. W., 1990), and L. Gold (Boulder) (Tuerk, C. and Gold, L, 1990). This technique is known as ‘in vitro selection, ‘in vitro evolution, or ‘SELEX’ (Systematic Evolution of Ligands by Exponential enrichment) all of which point to the evolutionary process of the selection. The functional molecules, called aptamers, can be separated from the non-functional molecules by a variety of separation techniques. This separation allows for the enrichment of the functional aptamers for the desired property. The functionalities of aptamers that have been discovered include the ability to bind to small organic molecules, to proteins that are known nucleotide binders, to proteins not known to bind nucleotides, and the alteration and development of ribozymes.
The starting pool of DNA for the technique is synthesized such that a random region in which each position may contain one of the four possible nucleotides. It is this region that brings in the complexity of the pool that generally ranges from 30 to 40 bases yielding up to 1.2×1016 to 1.2×1018 different sequences. This random region is flanked by two unique sequences that are used to amplify the DNA by PCR. Due to the tremendous number of different sequences that can be produced, which is much larger than the number of antibodies produced by mice, some of the sequences might have a desirable functionality. Very few of the aptamers present in the starting pool will have the desired functionality, so it is necessary to go through many successive selection and amplifications to obtain the desired aptamers (Klung and Famulok, 1994). Aptamers may consist of RNA, ssDNA, or dsDNA (FIG. 3) each of which follows similar procedures with unique steps pertaining to the type of aptamer being selected (Gold et al., 1995)